Cellular compartmentalization is a defining feature of life. Similar to eukaryotes, many prokaryotes compartmentalize their cytosol to carry out specialized metabolic reactions, prevent toxicity and store nutrients. However, prokaryotes rely on protein-based organelles instead of membrane-based compartmentalization strategies. A semipermeable protein shell creates a sequestered reaction space separated from the bulk cytosol. This compartmentalization can increase the local concentrations of enzymes and metabolites, prevent the leakage of toxic or volatile intermediates and create unique reaction environments. Protein organelles enable specialized biochemistry not possible without compartmentalization and have been implicated in carbon fixation, iron metabolism, nutrient utilization and stress resistance. Protein organelles are present in nearly all bacterial and archaeal phyla and can be found in many important human pathogens. Protein organelles have been suggested to enable pathogens to utilize alternative nutrients and withstand the human immune system. The two main classes of microbial protein organelles are encapsulin nanocompartments (20 to 50 nm) and bacterial microcompartments (40-500 nm). In both cases specialized enzymatic machinery is selectively encapsulated within self-assembling protein shells leading to unique catalytic capabilities. The overall goal of my laboratory is to explore and understand the functional diversity of protein organelles encoded in microbial genomes and to investigate their contribution towards human health and disease. We will initially prioritize protein organelles found in human-associated microbes. Hundreds of microbes encode uncharacterized protein organelle systems, some of which have been shown to contribute to microbial virulence. However, the molecular and physiological functions of most protein organelle systems have not been explored while their contributions towards human health and disease are poorly understood. The goals of this proposal are to (1) carry out a detailed mechanistic and structural analysis of the major classes of protein organelles, (2) determine how protein organelles contribute towards microbial stress resistance and detoxification and (3) investigate how protein organelles enable microbes to utilize alternative nutrient sources. Based on our expertise and pioneering investigations of encapsulin systems, we are uniquely positioned to successfully pursue the research described in this proposal. We will use a multifaceted approach including biochemistry, structural biology, microbiology and omics approaches to dissect the molecular mechanisms underlying protein organelle function and elucidate how these megadalton protein assemblies influence human health and disease. This information will be essential for exploring future therapeutic approaches aimed at disrupting protein organelle function to reduce microbial stress resistance and fitness and thus pathogenicity. The detailed characterization of protein organelles will also lay the groundwork for future protein organelle engineering with applications in biomanufacturing, biomedicine and bionanotechnology.